Post-Hypoglycemic hyperglycemia are highly relevant markers for stratification of glycemic variability and partial remission status of pediatric patients with new-onset type 1 diabetes

Aims To evaluate whether parameters of post-hypoglycemic hyperglycemia (PHH) correlated with glucose homeostasis during the first year after type 1 diabetes onset and helped to distinguish pediatric patients undergoing partial remission or not. Methods In the GLUREDIA (GLUcagon Response to hypoglycemia in children and adolescents with new-onset type 1 DIAbetes) study, longitudinal values of clinical parameters, continuous glucose monitoring metrics and residual β-cell secretion from children with new-onset type 1 diabetes were analyzed during the first year after disease onset. PHH parameters were calculated using an in-house algorithm. Correlations between PHH parameters (i.e., PHH frequency, PHH duration, PHH area under the curve [PHHAUC]) and glycemic homeostasis markers were studied using adjusted mixed-effects models. Results PHH parameters were strong markers to differentiate remitters from non-remitters with PHH/Hyperglycemia duration ratio being the most sensitive (ratio<0.02; sensitivity = 86% and specificity = 68%). PHHAUC moderately correlated with parameters of glucose homeostasis including TIR (R2 = 0.35, p-value < 0.05), coefficient of variation (R2 = 0.22, p-value < 0.05) and Insulin-Dose Adjusted A1c (IDAA1C) (R2 = 0.32, p-value < 0.05) and with residual β-cell secretion (R2 = 0.17, p-value < 0.05). Classification of patients into four previously described glucotypes independently validated PHH parameters as reliable markers of glucose homeostasis and improved the segregation of patients with intermediate values of IDAA1C and estimated C-peptide (CPEPEST). Finally, a combination of PHH parameters identified groups of patients with specific patterns of hypoglycemia. Conclusion PHH parameters are new minimal-invasive markers to discriminate remitters from non-remitters and evaluate glycemic homeostasis during the first year of type 1 diabetes. PHH parameters may also allow patient-targeted therapeutic management of hypoglycemic episodes.


Discussion
The first paragraph under the discussion is more of a literature for background than the beginning of discussion which usually has the major findings from the results.The inclusion criteria of experience with COVID-19 need further information as the frequency and how recent are likely to influence responses.

Conclusion
The Conclusion should specifically speak to aims of the study followed by implications of these findings.

See attached reviewer's comments
Reviewer's Responses to Questions Comments to the Author 1.Is the manuscript technically sound, and do the data support the conclusions?
The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions.Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes.The conclusions must be drawn appropriately based on the data presented.
Reviewer #1: No Reviewer #2: Yes 2. Has the statistical analysis been performed appropriately and rigorously?
Reviewer #1: Yes Reviewer #2: Yes 3. Have the authors made all data underlying the findings in their manuscript fully available?
The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file).The data should be provided as part of the manuscript or its supporting information or deposited to a public repository.For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available.If there are restrictions on publicly sharing data-e.g.participant privacy or use of data from a third party-those must be specified.
Reviewer #1: Yes Reviewer #2: Yes 4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous.Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Review Comments to the Author
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Reviewer #1: Abstract
The abstract should be restructured with the results section revised to include means percentages and standard deviations.
We thank the reviewer for the useful comments.Statistical values were added to the abstract as requested.Grammatical changes were also made in the manuscript.

What was the design of the study?
The present GLUREDIA study included data from the DIATAG multicentric and prospective study.Clarifications on this topic were added in the revised version of the manuscript in page 7.
The authors should indicate how the glycated hemoglobin was estimated?Glycated hemoglobin was measured from venous blood samples using high performance liquid chromatography.The reference cut-offs are those described by Tosoh (reference range 4-6% [20-42 mmol/mol]) (Tosoh G8; Tosoh, Tokyo, Japan).This precision was added in the revised version of the manuscript on page 7.
What is the definition of glucotypes and how did the authors come by the types?
Glucotypes correspond to daily glucose patterns.These were identified by our team during the 1 rst year of type 1 diabetes in pediatric patients in the context of the DIATAG study (Pollé et al., Diabetes Care 2022).Patients with new-onset type 1 diabetes distributed across these four glucotypes, progressively, from 3 months onwards.The glucotypes were identified using hierarchical clustering that included a combination of CGM metrics and clinical parameters (i.e., IDAA1C, HbA1C, IDD).This precision was already presented in the previous version of the manuscript (page 9).
The limitation should be added to the main text of the discussion.
Limitations were described as requested.
The length of the conclusion should also be reduced.
Conclusion length was reduced by more than 40% in the present manuscript (i.e., from 179 words to 103 words).

Reviewer #2: General Comment
This manuscript is a well thought of experiment that evaluates whether parameters of post-hypoglycemic hyperglycemia (PHH) correlated with glycemic homeostasis during the first year after type 1 diabetes onset and tries to use this to distinguish pediatric patients undergoing partial remission or not.
We thank the reviewer for this kind comment.
However, there are some specific comments that need attention.

Abstract;
There are several grammatical errors that authors need to correct.Moreover, there are abbreviations that need to be defined at first mention.Under the results, authors need to quote the estimates that indicate these PHH parameters can discriminate between remitters and non-remitters.
We have added the statistical data requested by Reviewer #1 in the abstract.Grammatical changes were also made in the manuscript.Also, the conclusion was revised as follows: "PHH parameters are new minimal-invasive markers to discriminate remitters from non-remitters and to assess glycemic homeostasis during the first year of type 1 diabetes that allow patient-specific therapeutic management".

Methodology
Under continuous glucose monitoring, authors indicate varied devices being used for this purpose but fail to indicate how these were standardized for the purpose of this study.This section requires further proof reading (see attached reviewer pdf).
As mentioned in our previous article that used a similar dataset, most patients were under Freestyle Libre CGM (i.e., >90%), and no sensor-specific pattern was observed within the principal component analysis 1 .This information was already mentioned in the first version of the manuscript (i.e., limitations on page 20).We nonetheless added additional precisions (i.e., PCA results from DIATAG study).
Authors do not justify the sample size used in the study.
The sample size was calculated at the initiation of DIATAG study by Pr.Annie Robert using PR occurrence as the major outcome.In recent studies, the partial remission period (i.e., IDAA1C < 9) has a mean of 9 months with a range from 2 to 24 months.Dividing 22 months by 6 provided an estimate of the standard deviation σ=3.7 months.With a sample size of 82 patients randomized in 2 balanced groups of 41 patients, the phase III clinical trial will have a power of 95% to detect an increase from 9 to 12 months in remission period, at the 0.05 statistical significance level.

Results
Authors should re-check the total number of outpatient visits in the first paragraph.There are sections under frequency of PHH which are better suited for methodology or discussion (see attached reviewer pdf).
We reviewed the total number of patients included in every analysis and corrected numbers accordingly in the manuscript.There was 244 outpatients visits with 52 that did not fully fulfilled inclusion criteria.Final analysis was performed on 192 visits from 66 patients.
Authors base conclusions on R2 values that are minimal with statistical significance and must discuss with this in mind..We think that adding such information may not improve the current manuscript.

Conclusion
The Conclusion should specifically speak to aims of the study followed by implications of these findings.
Conclusion was rewritten accordingly.
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